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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26 October 2015 to 04 February 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study conducted in compliance with OECD Guideline No. 406 without any deviation.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.6 (Skin Sensitisation)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
inspected on 23-24 April 2015 / signed on 23 October 2015
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
A scientific justification for completion of GPMT to fulfil the REACH chemical substance registration is attached to this ESR ("Attached Justification")

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
liquid
Details on test material:
- Physical state: Colourless liquid
- Storage Conditions: Stored in refrigerator (4 °C), darkness
Specific details on test material used for the study:
- Date received: 01 October 2015

In vivo test system

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan/Envigo, Kreuzelweg 53, 5961 NM Horst, The Netherlands.
- Age at study initiation: 3 or 4 weeks
- Housing: Animals were housed individually or in groups of 2 or 3 in polycarbonate containers.
- Diet: Food (SAFE, 106), ad libitum
- Water: Drinking water (tap water from public distribution system), ad libitum
- Acclimation period: 5 days
- Indication of any skin lesions: None

ENVIRONMENTAL CONDITIONS
- Temperature: 19–25 °C
- Humidity: 30-70 %
- Air changes: 10 changes per hour
- Photoperiod: 12 h dark / 12 h light

IN-LIFE DATES: 26 October 2015 to 04 February 2016

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal
Vehicle:
olive oil
Concentration / amount:
10% in olive oil
Day(s)/duration:
Day 0-6
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Route:
epicutaneous, occlusive
Vehicle:
other: liquid paraffin
Concentration / amount:
100% (undiluted)
Day(s)/duration:
Day 8-10
Adequacy of induction:
non-irritant substance, but skin pre-treated with 10% SDS
Challengeopen allclose all
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
unchanged (no vehicle)
Concentration / amount:
100% (undiluted)
Day(s)/duration:
Day 21-22
Adequacy of challenge:
highest non-irritant concentration
No.:
#2
Route:
epicutaneous, occlusive
Vehicle:
unchanged (no vehicle)
Concentration / amount:
100% (undiluted)
Day(s)/duration:
Days 28-29
Adequacy of challenge:
highest non-irritant concentration
No. of animals per dose:
10 and 5 animals for treatment and control groups, respectively
Details on study design:
PRELIMINARY STUDIES:
Determination by intradermal injection of the Maximal Non Necrotizing Concentration (MNNC): Two animals received a volume of 0.1 mL of the test item, on both sides of the spine, at 4 concentrations: 100% and diluted at 50, 20 and 10% in olive oil in order to determine the MNNC. A macroscopic evaluation of the cutaneous reactions was conducted 24 h after the injections.
Results: Necrosis or slight necrosis was evident at the 20%, 50% and 100% concentrations. No cutaneous reaction has been observed at the concentration of 10%. Therefore, 10% was selected for first induction in the main study.
Determination by topical application of the Pre-Maximal Non Irritant Concentration (Pre-MNIC): Test item was applied on the dorso-lumbar zone of two guinea pigs shorn beforehand, with occlusive dressing for 24 h, at 4 different concentrations: 100% and diluted at 50, 20 and 10% in liquid paraffin. A macroscopic evaluation of the cutaneous reactions was conducted 24 h after removal of the dressing.
Results: No cutaneous reactions were noted at any of the tested concentrations, 10, 20, 50 or 100%. In view of these results, the concentration selected was 100% for the 2nd induction and the MNIC determination began at the concentration of 100%.
Determination by topical application of the Maximal Non Irritant Concentration (MNIC): Three guinea pigs were treated according to the same treatment as animals from GROUP 1 (negative control) for the induction phase (i.e. olive oil and liquid paraffin). During the challenge phase, the animals were treated with the test item placed onto the selected treatment sites and covered with an occlusive dressing for a period of 24 h at 4 different concentrations: 100% and diluted at 50, 20 and 10% in liquid paraffin. A macroscopic evaluation of the cutaneous reactions was conducted 24 and 48 h after removal of the occlusive dressing.
Results: No cutaneous reactions were noted at any of the tested concentrations, 10, 20, 50 or 100%. In view of this result, the concentration selected was 100% (MNIC) for the challenge phase.

MAIN STUDY - FIRST TEST
A. INDUCTION EXPOSURE: INTRADERMAL
- No. of exposures: One
- Test groups: After shearing the scapular zone, three (3) pairs of intradermal injections (ID) of 0.1 mL were performed on the scapular zone in such a way as an injection on each pair is placed to either side of the spine as follows:
2 ID: Freund's Complete Adjuvant diluted at 50 % in olive oil
2 ID: test item at 10% in olive oil
2 ID: a test mixture in equal volumes v/v : Freund's Complete Adjuvant at 50% and the test item at 20% in olive oil
- Control group:
2 ID: Freund's Complete Adjuvant diluted at 50 % in olive oil
2 ID: olive oil
2 ID: a mixture with equal volumes v/v : - Freund's Complete Adjuvant at 50% and olive oil
- Site: Each side of the mid-line on scapular zone
- Exposure period: Day 0-6
- Duration: 7 days

Day 7: The scapular zone of all the animals in each group, shorn beforehand, was brushed with a solution of sodium lauryl sulfate at 10% in thick vaseline, in order to create a local irritation.

B. INDUCTION EXPOSURE: TOPICAL APPLICATION
- No. of exposures: One
- Exposure period: 48 h
Day 8: A topical application under occlusive dressing (25 mm x 25 mm non-woven swab of 4-layer patch from MEDISTOCK held in contact with the skin by means of 50 mm wide hypoallergenic micropore™ adhesive tape from 3M and Blenderm™ from 3M) for 48 h was performed on the injection sites of each animal.
- Test groups: 0.5 mL of the test item at 100%
- Control group: 0.5 mL of liquid paraffin
- Site: Same intradermally injected area of scapular zone
- Frequency of applications: Single application
Day 10: Occlusive dressing removal

REST PHASE
- The animals of both groups were left for 10 days.

C. CHALLENGE EXPOSURE - FIRST CHALLENGE
- No. of exposures: One
- Day(s) of challenge: Day 21
- Exposure period: 24 h
- Day 22: Occlusive dressing removal
- The experimental procedure of this phase was identical for both negative control and treated groups submitted to this experimentation: on the previously shorn dorso-lumbar zone, an application on the right flank, under occlusive dressing, was performed during 24 h:
- 1 sample cup (allergEAZE® clear patch test chamber - SmartPractice®) containing the test item at 100% (undiluted) (MNIC) and 1 sample cup (allergEAZE®clear patch test chamber SmartPractice®) containing liquid paraffin.
- Evaluation (h after removal of challenge patch): 24 and 48 h (Day 23 and 24)

REST PHASE
- The animals of both groups were left for 6 days.

D. CHALLENGE EXPOSURE - SECOND CHALLENGE
- No. of exposures: One
- Day(s) of challenge: Day 28
- Exposure period: 24 h
- Day 29: Occlusive dressing removal
- The experimental procedure of this phase was identical for both negative control and treated groups submitted to this experimentation: on the previously shorn dorso-lumbar zone, an application on the left flank, under occlusive dressing, was performed during 24 h:
- 1 sample cup (allergEAZE® clear patch test chamber - SmartPractice®) containing the test item at 100% (undiluted) (MNIC) and 1 sample cup (allergEAZE®clear patch test chamber SmartPractice®) containing liquid paraffin.
- Evaluation (h after removal of challenge patch): 24 and 48 h (Day 30 and 31)

Due to equivocal borderline results, under the experimental conditions, a 2nd test was conducted with 5 new control animals and 10 treated animals in the same experimental conditions.

MAIN STUDY - SECOND TEST
A. INDUCTION EXPOSURE: INTRADERMAL
- No. of exposures: One
- Test groups: After shearing the scapular zone, three (3) pairs of intradermal injections (ID) of 0.1 mL were performed on the scapular zone in such a way as an injection on each pair is placed to either side of the spine as follows:
2 ID: Freund's Complete Adjuvant diluted at 50 % in olive oil
2 ID: test item at 10% in olive oil
2 ID: a test mixture in equal volumes v/v : Freund's Complete Adjuvant at 50% and the test item at 20% in olive oil
- Control group:
2 ID: Freund's Complete Adjuvant diluted at 50 % in olive oil
2 ID: olive oil
2 ID: a mixture with equal volumes v/v : - Freund's Complete Adjuvant at 50% and olive oil
- Site: Each side of the mid-line on scapular zone
- Exposure period: Day 0-6
- Duration: 7 days

Day 7: The scapular zone of all the animals in each group, shorn beforehand, was brushed with a solution of sodium lauryl sulfate at 10% in thick vaseline, in order to create a local irritation.

B. INDUCTION EXPOSURE: TOPICAL APPLICATION
- No. of exposures: One
- Exposure period: 48 h
Day 8: A topical application under occlusive dressing (25 mm x 25 mm non-woven swab of 4-layer patch from MEDISTOCK held in contact with the skin by means of 50 mm wide hypoallergenic micropore™ adhesive tape from 3M and Blenderm™ from 3M) for 48 h was performed on the injection sites of each animal.
- Test groups: 0.5 mL of the test item at 100%
- Control group: 0.5 mL of liquid paraffin
- Site: Same intradermally injected area of scapular zone
- Frequency of applications: Single application
Day 10: Occlusive dressing removal

REST PHASE
- The animals of both groups were left for 10 days.

C. CHALLENGE EXPOSURE - FIRST CHALLENGE
- No. of exposures: One
- Day(s) of challenge: Day 21
- Exposure period: 24 h
- Day 22: Occlusive dressing removal
- The experimental procedure of this phase was identical for both negative control and treated groups submitted to this experimentation: on the previously shorn dorso-lumbar zone, an application on the right flank, under occlusive dressing, was performed during 24 h:
- 1 sample cup (allergEAZE® clear patch test chamber - SmartPractice®) containing the test item at 100% (undiluted) (MNIC) and 1 sample cup (allergEAZE®clear patch test chamber SmartPractice®) containing liquid paraffin.
- Evaluation (h after removal of challenge patch): 24 and 48 h (Day 23 and 24)
Challenge controls:
Yes, 5 animals, with challenge at 100%.
Positive control substance(s):
yes
Remarks:
α-Hexylcinnamaldehyde

Results and discussion

Positive control results:
α-Hexylcinnamaldehyde induced skin sensitisation indicating the validity of the study (Study No.: SMK-2014-002; SMK-2015-001; SMK-2015-002).

In vivo (non-LLNA)

Resultsopen allclose all
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
100% test item
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
no cutaneous allergic reaction was noted
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
100% test item
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
no cutaneous allergic reaction was noted
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
100% test item
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
no cutaneous allergic reaction was noted
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
100% test item
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
no cutaneous allergic reaction was noted
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
12.5 %
No. with + reactions:
8
Total no. in group:
10
Remarks on result:
positive indication of skin sensitisation
Remarks:
Not performed within this test, results came from historical control data
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
12.5%
No. with + reactions:
4
Total no. in group:
10
Remarks on result:
positive indication of skin sensitisation
Remarks:
Not performed within this test, results came from historical control data

Any other information on results incl. tables

First test

Induction phase Group 2-Treated group: No cutaneous reaction after the first induction. Dryness of the skin was noted in all animals (10/10) after the second induction.

Induction phase Group 1-Control group: No cutaneous reaction was recorded during the induction phase.

First challenge Groups 1 & 2:

In the treated group (treatment dose of 100%), a discrete to well defined erythema was recorded in 40% (4/10) of the animals, 24 hours after the challenge phase. No cutaneous allergic reaction was noted 48 hours after the challenge phase.

In the control group (associated with the treatment dose of 100%), no cutaneous reaction was noted after the challenge phase (0/5).

A discrete erythema was recorded in 10% (1/10) of animals from the treated group 24 hours after the challenge phase, on the treated area with liquid paraffin (control item). The vehicle created an equivocal result in one treated animal challenge with vehicle only.

No cutaneous reaction was recorded in animals (0/5) from the control group after the challenge phase, on the treated area with liquid paraffin (control item).

To clarify the results obtained with the test item, a second challenge phase was performed with the test item under the same experimental conditions after a rest phase of 6 days.

Second challenge Groups1 & 2:

Due to the equivocal result, the same animals were rechallenged.

In the treated group (treatment dose of 100%), a discrete to intense erythema was recorded in 40% (4/10) of the animals, 24 hours after the rechallenge phase. No cutaneous allergic reaction was noted 48 hours after the rechallenge phase.

In the control group (associated with the treatment dose of 100%), a moderate erythema was recorded in 40% (2/5) of the animals 24 hours after the rechallenge phase. No cutaneous reaction was noted 48 hours after the rechallenge phase.

A discrete erythema was recorded in 10% (1/10) of animals from the treated group 24 hours after the rechallenge phase, on the treated area with liquid paraffin (control item).

No cutaneous reaction was recorded in animals (0/5) from the control group after the challenge phase, on the treated area with liquid paraffin (control item).

It therefore appears clear that the vehicle is having some impact on the results conducted in the first test. Due to the equivocal results, under these experimental conditions, a 2nd test was conducted with 5 new control animals and 10 treated animals in the same experimental conditions.

 

Second test

Induction phase Group 2-Treated group: Discrete erythema was noted in four animals (4/10) and no cutaneous reaction was noted in the other animals (6/10) after the first induction. Dryness of the skin was noted in all animals (10/10) after the second induction.

Induction phase Group 1-Control group: No cutaneous reaction was noted during the first induction. Dryness of the skin was noted in three animals (3/5) after the second induction.

Challenge phase Groups 1& 2:

In the treated group (treatment dose of 100%), no cutaneous allergic reaction was noted 24 and 48 hours after the challenge phase (0/10).

In the control group (associated with the treatment dose of 100%), no cutaneous reaction was noted 24 and 48 hours after the challenge phase (0/5).

No cutaneous reaction was recorded in animals (0/10) from the treated group after the challenge phase, on the treated area with liquid paraffin (control item).

No cutaneous reaction was recorded in animals (0/5) from the control group after the challenge phase, on the treated area with liquid paraffin (control item).

 

Weight evolution: No abnormality was recorded in the body weight gain of both groups.

Mortality: No mortality was registered during the main study.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Under the experimental conditions, the test item is not classified as a skin sensitizer, according to Regulation (EC) No. 1272/2008 (CLP) and to GHS.
Executive summary:

In a Magnusson & Kligman maximisation study (GPMT) performed according to OECD Guideline 406 and in compliance with GLP, test item was assessed for skin sensitization in female Dunkin-Hartley Guinea pigs. After induction (intradermal injection at 10% and topical application at 100%) of 10 Guinea pigs of treated group with the test item and a 10-day rest phase, the challenge phase, under occlusive dressing for 24 hours, consisted of a single topical application of the test item at 100% and of a negative control (liquid paraffin). Control group of 5 females treated in similar manner. The test concentrations for the main study were determined from a preliminary study.

 

First test:

In the treated group (treatment dose of 100%), a discrete to well defined erythema was recorded in 40% (4/10) of the animals, 24 hours after the challenge phase. No cutaneous allergic reaction was noted 48 hours after the challenge phase.

In the control group (associated with the treatment dose of 100%), no cutaneous reaction was noted after the challenge phase (0/5).

A discrete erythema was recorded in 10% (1/10) of animals from the treated group 24 hours after the challenge phase, on the treated area with liquid paraffin (control item). The vehicle created an equivocal result in one treated animal challenge with vehicle only.

No cutaneous reaction was recorded in animals (0/5) from the control group after the challenge phase, on the treated area with liquid paraffin (control item).

To clarify the results obtained with the test item, a second challenge phase was performed with the test item under the same experimental conditions after a rest phase of 6 days. Due to the equivocal result, the same animals were rechallenged.

In the treated group (treatment dose of 100%), a discrete to intense erythema was recorded in 40% (4/10) of the animals, 24 hours after the rechallenge phase. No cutaneous allergic reaction was noted 48 hours after the rechallenge phase.

In the control group (associated with the treatment dose of 100%), a moderate erythema was recorded in 40% (2/5) of the animals 24 hours after the rechallenge phase. No cutaneous reaction was noted 48 hours after the rechallenge phase.

A discrete erythema was recorded in 10% (1/10) of animals from the treated group 24 hours after the rechallenge phase, on the treated area with liquid paraffin (control item).

No cutaneous reaction was recorded in animals (0/5) from the control group after the challenge phase, on the treated area with liquid paraffin (control item).

It therefore appears clear that the vehicle is having some impact on the results conducted in the first test. Due to the equivocal results, under these experimental conditions, a 2nd test was conducted with 5 new control animals and 10 treated animals in the same experimental conditions.

Second test:

In the treated group (treatment dose of 100%), no cutaneous allergic reaction was noted 24 and 48 hours after the challenge phase (0/10).

In the control group (associated with the treatment dose of 100%), no cutaneous reaction was noted 24 and 48 hours after the challenge phase (0/5).

No cutaneous reaction was recorded in animals (0/10) from the treated group after the challenge phase, on the treated area with liquid paraffin (control item).

No cutaneous reaction was recorded in animals (0/5) from the control group after the challenge phase, on the treated area with liquid paraffin (control item).

 

Under the experimental conditions, the test item is not classified as a skin sensitizer, according to Regulation (EC) No. 1272/2008 (CLP) and to GHS.